The long form of tRNase Z (tRNase ZL) in eukaryotes endonucleolytically removes 3' trailers from pre-tRNAs, leaving a3'-OH prepared for CCA addition. tRNase Z reaction is thus central to tRNA maturation and function in translation. tRNase Z enzymology and structural biophysics will be analyzed as follows: (Aim 1) Do novel putative pathogenesis-related mitochondrial tRNA mutations change tRNase Z processing and tRNA structure? (Aim 2) Mutations in H. sapiens tRNase ZL associated with cardiac hypertrophy affect pre- tRNA processing kinetics. (Aim 3) A. Conserved sequence elements preceding and within the flexible tether affect tRNase Z enzyme activity and flexibility; B. Splicing isoforms ofH. sapiens tRNase ZL affect protein flexibility. Aim 4. Is the tRNase Z - mature tRNA 3'-end CCA anti-determinant localized to a specific residue or region? tRNase Z enzymology and structural biophysics provides an effective platform for providing research laboratory training to a new generation of students at a primarily undergraduate, minority-serving institution to enter the pipeline for PhD-level training in biomedical research. The project will be supported by three effective collaborations.